Splice-region antisense composition and method

ABSTRACT

Antisense compositions targeted against an mRNA sequence coding for a selected protein, at a region having its 5′ end from 1 to about 25 base pairs downstream of a normal splice acceptor junction in the preprocessed mRNA, are disclosed. The antisense compound is RNase-inactive, and is a phosphorodiamidate-linked morpholino oligonucleotide containing uncharged phosphorodiamidate linkages interspersed with cationic phosphorodiamidate linkages. Such targeting is effective to inhibit natural mRNA splice processing, produce splice variant mRNAs, and inhibit normal expression of the protein.

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/893,086, filed Jul. 16, 2004, now pending, which is acontinuation of U.S. patent application Ser. No. 09/848,868, filed May4, 2001, now U.S. Pat. No. 6,784,291, which claims priority to U.S.provisional application Ser. No. 60/202,376, filed May 4, 2000, all ofwhich are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to therapeutic compositions and methodsfor inhibiting expression of full-length proteins in cells, and inparticular to antisense compositions targeted against an mRNA sequencehaving its 5′ end 1 to about 25 base pairs downstream of a normal spliceacceptor junction in a preprocessed mRNA. Such targeting is effective toinhibit natural mRNA splice processing and produce splice variant mRNAs.

BACKGROUND OF THE INVENTION

Inhibition of protein expression by antisense targeting of DNA or RNAcoding for the protein has been the subject of extensive study. Manyreported procedures have employed phosphorothioate-linkedoligonucleotides, which are charged, nuclease-resistant analogs ofnative DNA. The antisense mechanism involved is based on the activationof RNase, which cleaves the target nucleic acid to which the oligomer isbound. While these compounds have shown high activity, they also tend toshow high levels of side effects, i.e. by cleavage of non-target RNA orby non-antisense mechanisms, such as nonspecific binding to proteins.

Another class of antisense oligomers, termed RNase-inactive, do notpromote cleavage of bound RNA and are believed to act by stericallyblocking the molecular machinery from transcribing, processing, ortranslating the target sequence. While these compounds tend to producefewer side reactions, such as nonselective cleavage, thanphosphorothioate oligomers, it has generally been necessary to targetspecific regions of RNA, such as the AUG start codon, for successfulinhibition.

More recently, targeting of the splice acceptor junction of nuclear(unspliced) RNA by RNase-inactive oligomers has been reported. Kole andDominski (U.S. Pat. No. 5,665,593) reported suppression of missplicingof β-globin RNA, in order to combat variants of β-thalassemia whichresult from such aberrant splicing. In this case, the aberrant splicejunction was targeted, to direct splicing back to the normal site. RVGiles et al., Antisense & Nucleic Acid Drug Dev. 9:213-220 (1999),targeted a splice junction to induce missplicing of c-myc mRNA. In eachof these cases, the region targeted is still somewhat restricted, inthat the antisense oligomer spans the intron/exon splice junction of thepre-mRNA. Due to the advantages accorded by the use of uncharged,RNase-inactive oligonucleotides, a demonstration of further flexibilityin targeting would be quite useful.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an antisense compound, and acorresponding method of inhibiting normal splicing of preprocessed RNAin a eukaryotic cell, by contacting the cell with such an antisensecompound. The compound is characterized by:

(a1): a morpholino backbone composed of morpholino subunits that arelinked by uncharged phosphorodiamidate linkages interspersed with atleast two and up to half positively charged phosphorodiamidate linkages;

(a2): a base-sequence length of between 12 and 25 nucleotide bases; and

(a3): a base sequence that is complementary to a target region of aselected preprocessed mRNA coding for a selected protein, where the 5′end of the target region is 1-25 bases downstream of a normal spliceacceptor site in the preprocessed mRNA, and having the properties that:

(b1): the compound is taken up by eukaryotic cells;

(b2): the compound hybridizes to the target region of preprocessed mRNAin such cells, and

(b3): the compound so hybridized to the target pre-mRNA preventssplicing at the normal acceptor splice site, such that the splicemechanism proceeds to a downstream splice acceptor site in thepreprocessed mRNA, producing a splice variant processed mRNA with atruncated coding sequence.

In more specific embodiments, the 5′ end of the target region is 2-20bases, or 2-15 bases, downstream of the normal splice acceptor site. Thelength of the targeting compound is preferably about 15 to 20 nucleotidebases.

In one embodiment, the morpholino subunits are linked byphosphorodiamidate linkages, in accordance with the structure:

where Y₁═O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X for the uncharged linkages is alkyl, alkoxy,thioalkoxy, or an alkyl amino of the form wherein NR₂, where each R isindependently hydrogen or methyl, and for the positively chargedlinkages, X is 1-piperazine.

When the downstream splice acceptor site is a whole multiple of threebases downstream of the normal splice acceptor site, the splice variantmRNA has a coding sequence in frame with that of the processed mRNA whenit is normally spliced.

In selected embodiments, the selected protein has multiple distinctbinding regions, as in most transcription factors, and the truncatedcoding sequence codes for a variant protein in which one such bindingregion is disabled. Preferably, the variant protein is a dominantnegative protein. One example is human c-myc, where the variant proteinis an N-terminal truncated c-myc. In this embodiment, the antisensecompound employed has a base sequence selected from the group consistingof SEQ ID NO: 3 and SEQ ID NOs: 13 through 28 herein. The variantprotein may also be a C-terminal altered c-myc, in which case theantisense compound employed can have the base sequence SEQ ID NO: 29.

In additional exemplary embodiments, the selected protein and thecorresponding antisense base sequence(s) targeting its pre-mRNA areselected from the group consisting of:

(a) human chorionic gonadotropin, β subunit:

(b) human androgen receptor:

(c) human c-myc:

(d) human p53:

(e) human abl: and

(f) HIV-1 rev.

In another aspect, the invention includes a method of inhibiting normalsplicing of mRNA in a eukaryotic cell, by contacting the cell with thean antisense compound of the type described above.

These and other objects and features of the present invention willbecome more fully apparent when the following detailed description ofthe invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show several preferred subunits having 5-atom (A), six-atom(B) and seven-atom (C-E) linking groups suitable for forming polymers,and

FIGS. 2A-A to 2E-E show the repeating subunit segment of exemplarymorpholino oligonucleotides, designated A-A through E-E, constructedusing subunits A-E, respectively, of FIG. 1. FIGS. 2F and 2G showmorpholino subunits linked with uncharged and positively chargedphosphorodiamidate linkages, respectively.

FIG. 3 shows the synthetic steps to produce subunits used to producemorpholino oligomers containing the (1-piperazino) phosphinylideneoxycationic linkage as shown in FIG. 2G.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

“Antisense” refers to an oligomer having a sequence of nucleotide basesand a subunit-to-subunit backbone that allows the antisense oligomer tohybridize to a target sequence in an RNA by Watson-Crick base pairing,to form an RNA:oligomer heteroduplex within the target sequence,typically with an mRNA. The oligomer may have exact sequencecomplementarity to the target sequence or near complementarity. Theseantisense oligomers may block or inhibit translation of the mRNA, and/ormodify the processing of an mRNA to produce a splice variant of themRNA.

As used herein, the terms “compound”, “agent”, “oligomer” and“oligonucleotide” may be used interchangeably with respect to theantisense oligonucleotides of the invention.

A “subunit” of an oligonucleotide analog refers to one nucleotide (ornucleotide analog) unit of the analog. The term may refer to thenucleotide unit with or without the attached intersubunit linkage,although, when referring to a “charged subunit”, the charge typicallyresides within the intersubunit linkage (e.g. a phosphate orphosphorothioate linkage).

As used herein, a “morpholino oligomer” refers to a polymericoligonucleotide analog molecule having a backbone which supports basescapable of hydrogen bonding to typical polynucleotides, wherein thepolymer lacks a pentose sugar backbone moiety, and more specifically aribose backbone linked by phosphodiester bonds which is typical ofnucleotides and nucleosides, but instead contains a ring nitrogen withcoupling through the ring nitrogen. A preferred “morpholino”oligonucleotide is composed of morpholino subunit structures of the formshown in FIG. 2B-B, where (i) the structures are linked together byphosphorous-containing linkages, one to three atoms long, joining themorpholino nitrogen of one subunit to the 5′ exocyclic carbon of anadjacent subunit, and (ii) P_(i) and P_(j) are purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide. Exemplary structures forantisense oligonucleotides for use in the invention include themorpholino subunit types shown in FIGS. 1A-E, with the linkages shown inFIGS. 2A-A to 2E-E.

A “subunit” of an oligonucleotide or oligonucleotide analog refers toone nucleotide (or nucleotide analog) unit of the oligomer. The term mayrefer to the nucleotide unit with or without the attached intersubunitlinkage, although, when referring to a “charged subunit”, the chargetypically resides within the intersubunit linkage (e.g. a phosphate orphosphorothioate linkage).

A “morpholino oligomer” is an oligonucleotide analog composed ofmorpholino subunit structures of the form shown in FIGS. 1 A-E, where(i) the structures are linked together by phosphorus-containinglinkages, one to three atoms long, joining the morpholino nitrogen ofone subunit to the 5′ exocyclic carbon of an adjacent subunit, and (ii)P_(i) and P_(j) are purine or pyrimidine base-pairing moieties effectiveto bind, by base-specific hydrogen bonding, to a base in apolynucleotide. The purine or pyrimidine base-pairing moiety istypically adenine, cytosine, guanine, uracil or thymine. The synthesis,structures, and binding characteristics of morpholino oligomers aredetailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,5,166,315, 5,521,063, and 5,506,337, all of which are incorporatedherein by reference.

The subunit and linkage shown in FIG. 1B are used for six-atomrepeating-unit backbones, as shown in FIG. 2B (where the six atomsinclude: a morpholino nitrogen, the connected phosphorus atom, the atom(usually oxygen) linking the phosphorus atom to the 5′ exocyclic carbon,the 5′ exocyclic carbon, and two carbon atoms of the next morpholinoring). In these structures, the atom Y₁ linking the 5′ exocyclicmorpholino carbon to the phosphorus group may be sulfur, nitrogen,carbon or, preferably, oxygen. The X moiety pendant from the phosphorusis any stable group which does not interfere with base-specific hydrogenbonding. Preferred X groups include fluoro, alkyl, alkoxy, thioalkoxy,and alkyl amino, including cyclic amines, all of which can be variouslysubstituted, as long as base-specific bonding is not disrupted. Alkyl,alkoxy and thioalkoxy preferably include 1-6 carbon atoms. Alkyl aminopreferably refers to lower alkyl (C₁ to C₆) substitution, and cyclicamines are preferably 5- to 7-membered nitrogen heterocycles optionallycontaining 1-2 additional heteroatoms selected from oxygen, nitrogen,and sulfur. Z is sulfur or oxygen, and is preferably oxygen.

The morpholino oligomer of the present invention is aphosphorodiamidate-linked morpholino oligomer, referred to herein as aPMO, and includes uncharged phosphorodiamidate linkages interspersedwith positively charged phosphorodiamidate linkages. Such oligomers arecomposed of morpholino subunit structures such as shown in FIG. 1B,where X═NH2, NHR, or NR2 (where R is lower alkyl, preferably methyl)(See FIG. 2F), Y═O, and Z=O, and Pi and Pj are purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide, for the uncharged linkages, andthe same structure, but where X-1-piperazine for the charged linkages(see FIG. 2G). The uncharged linkages may also have the structure shownin FIG. 1B, but where X=lower alkoxy, such as methoxy or ethoxy, Y═NH orNR, where R is lower alkyl, and Z=O.

A “nuclease-resistant” oligomeric molecule (oligomer) is one whosebackbone is not susceptible to nuclease cleavage.

A substantially uncharged, phosphorus containing backbone in anoligonucleotide analog is one in which a majority of the subunitlinkages, e.g., between 50-100%, are uncharged at physiological pH, andcontain a single phosphorous atom. The analog contains between 12 and 40subunits, typically about 15-25 subunits, and preferably about 18 to 25subunits. The analog may have exact sequence complementarity to thetarget sequence or near complementarity, as defined below.

As used herein, an oligonucleotide or antisense oligomer “specificallyhybridizes” to a target polynucleotide if the oligomer hybridizes to thetarget under physiological conditions, with a Tm substantially greaterthan 37° C., preferably at least 50° C., and typically 60° C.-80° C. orhigher. Such hybridization preferably corresponds to stringenthybridization conditions, selected to be about 101C, and preferablyabout 50° C. lower than the thermal melting point (T[m]) for thespecific sequence at a defined ionic strength and pH. At a given ionicstrength and pH, the T[m] is the temperature at which 50% of a targetsequence hybridizes to a complementary polynucleotide.

Polynucleotides are described as “complementary” to one another whenhybridization occurs in an antiparallel configuration between twosingle-stranded polynucleotides. A double-stranded polynucleotide can be“complementary” to another polynucleotide, if hybridization can occurbetween one of the strands of the first polynucleotide and the second.Complementarity (the degree that one polynucleotide is complementarywith another) is quantifiable in terms of the proportion of bases inopposing strands that are expected to form hydrogen bonds with eachother, according to generally accepted base-pairing rules.

A “RNase-inactive” or “RNase-incompetent” oligonucleotide oroligonucleotide analog is one which acts via an RNase-independentmechanism, unlike RNase-active oligonucleotides, such asphosphorothioates. They are believed to function by sterically blockingtarget RNA formation, nucleocytoplasmic transport or translation, andare thus also referred to as “steric blockers”. This class includes, forexample, methylphosphonates, morpholino oligonucleotides, as describedherein, peptide nucleic acids (PNA's), and 2′-O-allyl or 2′-O-alkylmodified oligonucleotides.

In a “peptide nucleic acid”, the deoxyribose phosphate units of anoligonucleotide backbone are replaced with polyamide linkages. Properbackbone spacing is attained by the use of 2-aminoethyl glycine units,with a nucleotide base attached to each 2-amino group via amethylenecarbonyl group.

A “2′-O-allyl (or alkyl) modified oligonucleotide” is anoligoribonucleotide in which the 2′ hydroxyl is converted to an alkyl oralkyl ether. The alkyl ether is typically a methyl ether.

“Alkyl” refers to a fully saturated acyclic monovalent radicalcontaining carbon and hydrogen, which may be branched or a straightchain. Examples of alkyl groups are methyl, ethyl, n-butyl, t-butyl,n-heptyl, and isopropyl. “Lower alkyl” refers to an alkyl radical of oneto six carbon atoms, and preferably one to four carbon atoms, asexemplified by methyl, ethyl, isopropyl, n-butyl, isobutyl, and t-butyl.

A “truncated” protein or coding sequence has some portion of the normalprotein or sequence removed from one or the other terminus, from aninternal region, or a combination of the above.

An amino-truncated (N-truncated) or carboxy-truncated (C-truncated)protein is one having an abnormal or deleted amine terminus or carboxyterminus, respectively, arising from translation of a splice variantmRNA.

II. Antisense Compounds

In accordance with the present invention, it has been discovered that anantisense compound having from 12 to 25 nucleotides, including atargeting base sequence that is complementary to a target region of aselected preprocessed mRNA coding for a selected protein, where the 5′end of the target region is 1 to 25 bases downstream, preferably 2 to 20bases downstream, and more preferably 2 to 15 bases downstream, of anormal splice acceptor site in the preprocessed mRNA, is effective toinhibit splicing at the normal splice acceptor site and thus producesplice variant mRNA, leading to truncated or otherwise aberrant versionsof the selected protein upon translation. Advantages of this strategyare set forth below.

The antisense compound employed in the present invention is one thatdoes not activate RNase H. RNase-H active oligomers, of whichphosphorothioate oligonucleotides are the most prominent example,operate primarily by a mechanism in which the target mRNA is cleaved.RNase-incompetent oligomers, on the other hand, are believed to act by asteric blocking mechanism. Such compounds include morpholino oligomers,PNA's (peptide nucleic acids), methylphosphonates, and 2′-O-alkyl or-allyl modified oligonucleotides, all of which are known in the art. Thepreferred antisense oligomers (compounds) of the present invention aremorpholino oligomers, which are composed of morpholino subunits of theform shown in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,5,166,315, 5,521,063, and 5,506,337, all of which are incorporatedherein by reference. The synthesis, structures, and bindingcharacteristics of morpholino oligomers are detailed in these patents.In a morpholino oligomer, (i) the morpholino groups are linked togetherby either uncharged or cationic phosphorus-containing linkages, one tothree atoms long, joining the morpholino nitrogen of one subunit to the5′ exocyclic carbon of an adjacent subunit, and (ii) the base attachedto the morpholino group is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide. The purine or pyrimidine base-pairing moiety istypically adenine, cytosine, guanine, uracil, thymine, or inosine.Preparation of such oligomers is described in detail in U.S. Pat. No.5,185,444 (Summerton and Weller, 1993), which is hereby incorporated byreference in its entirety. As shown in the reference, several types ofnonionic linkages may be used to construct a morpholino backbone.

Such morpholino oligomers have shown high binding affinity for RNAtargets, and the uncharged backbone favors uptake into cells and reducesnon-specific binding interactions, relative to charged analogs such asphosphorothioates. They have been shown to provide significantlyimproved activity and selectivity in inhibiting translation of targetedsequences in comparison to phosphorothioate oligonucleotides. See, forexample, Summerton et al., Antisense & Nucleic Acid Drug Dev.7(2):63-70, April 1997. The morpholino oligomers have very high nucleaseresistance and good water solubility, making them good candidates for invivo use. Efficient uptake by cells in vivo is demonstrated in co-ownedand copending application Ser. No. 09/493,427 and the corresponding PCTPubn. No. WO 0044897. As described therein, morpholino oligonucleotideshaving phosphoramidate linkages formed heteroduplexes with target RNA,which are protected in this duplex state from nuclease degradation. Sucha duplex is expelled from the cell, and the target RNA can later bedetected in a body fluid sample from the subject. These resultsdemonstrated that the morpholino oligomers (i) migrate to and entercells in the body and (ii) bind with high affinity, via Watson-Crickbase-pairing, to target nucleic acid regions.

Exemplary backbone structures for antisense oligonucleotides of theinvention include the β-morpholino subunit types shown in FIG. 1A-E, asdescribed above. It will be appreciated that a polynucleotide maycontain more than one linkage type.

A preferred morpholino oligomer is a phosphorodiamidate-linkedmorpholino oligomer, referred to herein as a PMO. Such oligomers arecomposed of morpholino subunit structures such as shown in FIG. 2B-B,where X═NH₂, NHR, or NR₂ (where R is lower alkyl, preferably methyl),Y═O, and Z=O, and P_(i) and P_(j) are purine or pyrimidine base-pairingmoieties effective to bind, by base-specific hydrogen bonding, to a basein a polynucleotide. Also preferred are morpholino oligomers where thephosphordiamidate linkages are a mixture of uncharged linkages as shownin FIG. 2F and cationic linkages as shown in FIG. 2G where, in FIG.2B-B, X=1-piperazino. Also preferred are structures having an alternatephosphorodiamidate linkage, where, in FIG. 2B-B, X=lower alkoxy, such asmethoxy or ethoxy, Y═NH or NR, where R is lower alkyl, and Z=O. As noteabove, the oligomer backbone in the present invention is formed ofuncharged phosphorodiamidate linkages interspersed with cationicphosphorodiamidate linkages. Exemplary linkages are shown in Fig. FIG.2B-B, where X NH₂, NHR, or NR₂ (where R is lower alkyl, preferablymethyl), Y═O, and Z=O, and P_(i) and P_(j) are purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide, for the uncharged linkages(shown in FIG. 2F), and where X=1-piperazino for the charged linkages(shown in FIG. 2G). The uncharged linkages may alternatively have thesame FIG. 2B-B structure, where, in FIG. 2B-B, X=lower alkoxy, such asmethoxy or ethoxy, Y═NH or NR, where R is lower alkyl, and Z=O.

The solubility of the antisense compound, and the ability of thecompound to resist precipitation on storage in solution, can be furtherenhanced by derivatizing the oligomer with a solubilizing moiety, suchas a hydrophilic oligomer, or a charged moiety, such as a charged aminoacid or organic acid. The moiety may be any biocompatible hydrophilic orcharged moiety that can be coupled to the antisense compound and thatdoes not interfere with compound binding to the target sequence. Themoiety can be chemically attached to the antisense compound, e.g., atits 5′ end, by well-known derivatization methods. One preferred moietyis a defined length oligo ethylene glycol moiety, such astriethyleneglycol, coupled covalently to the 5′ end of the antisensecompound through a carbonate linkage, via a piperazine linking groupforming a carbamate linkage with triethyleneglycol, where the secondpiperazine nitrogen is coupled to the 5′-end phosphorodiamidate linkageof the antisense. Alternatively, or in addition, the compound may bedesigned to include one a small number of charged backbone linkages,such as a phosphodiester linkage, preferably near one of the ends of thecompound. The added moiety is preferably effective to enhance solubilityof the compound to at least about 30 mgs/ml, preferably at least 50mgs/ml in aqueous medium.

The compound is designed to hybridize to the target sequence underphysiological conditions with a T_(m) substantially greater than 37° C.,e.g., at least 50° C. and preferably 60° C.-80° C. Although the compoundis not necessarily 100% complementary to the target sequence, it iseffective to stably and specifically bind to the target sequence suchthat expression of the target sequence, is modulated. The appropriatelength of the oligomer to allow stable, effective binding combined withgood specificity is about 8 to 40 nucleotide base units, and preferablyabout 12-25 base units. Mismatches, if present, are less destabilizingtoward the end regions of the hybrid duplex than in the middle. Oligomerbases that allow degenerate base pairing with target bases are alsocontemplated, assuming base-pair specificity with the target ismaintained.

III. Selection of Target Sequences

A. RNA Splicing: Background

The processing of nuclear RNA following transcription is observed invirtually all living cells. The mammalian genome contains genes thatmake transcripts of approximately 16,000 bases in length containing 7 to8 exons. The process of splicing reduces the length of the mRNA to anaverage of 2,200 bases. The initial transcript is referred to asheterologous nuclear RNA (hnRNA) or pre-mRNA. Processing of hnRNAinvolves an aggregate of approximately 20 proteins, referred tocollectively as the spliceosome, which carries out splicing andtransport of mRNA from the nucleus. The spliceosome does not appear toscan from a common direction for all transcripts; introns may be removedin a reproducible order but not in a directional order. For example,introns 3 and 4 may be removed first, followed by removal of introns 2and 5, followed by removal of introns 1 and 6. The order of intronremoval is not predictable a priori of observation. The sequencerecognition for processing is small, suggesting that errors ormultiplicity of processing sites can be anticipated, and, in fact, asmore genes are investigated, more variation in processing of hnRNA hasbeen observed.

In preprocessed mRNA, the two-base sequence motifs at exon/intronjunctions are invariant. The upstream (5′) splice donor (SD) junction isof the form exon-/GT-intron, while the downstream (3′) splice acceptor(SA) junction is of the form intron-AG/exon. The flanking bases are notinvariant, however, the base immediately upstream of the splice acceptorAG sequence is C about 80% of the time.

The current understanding of intron sequence recognition is as follows:5′-exonA₍₆₄₎G₍₇₃₎/G ₍₁₀₀₎ T ₍₁₀₀₎A₍₆₂₎A₍₆₈₎G₍₈₄₎T₍₆₃₎...TACTA AC...C₍₈₀₎ A ₍₁₀₀₎ G ₍₁₀₀₎/exon-NN...-3′      splice donor(SD)                                splice acceptor (SA)

The numbers in parenthesis represent the approximate (when <100%)percent utilization of a base at a site. The bold A in the middle of theintron is the site (branch point) at which the G from the splice donorforms a branched 2′-5′-structure referred to as the lariat. The sequenceindicated (TACTAAC) is the consensus observed in yeast. The consensussequence in mammalian cells is PyNPy₍₈₀₎Py₍₈₇₎Pu₍₇₅₎APy₍₇₅₎, where Pyrepresents a pyrimidine (U/T or C) and Pu represents a purine (A or G).The A at the branch point is invariant, and is typically found about 12to 50 bases upstream of the AG site. A pyrimidine-rich region (not shownabove) is also generally found near the 3′ end of the intron, about10-15 bases upstream of the AG site.

Once the spliceosome forms the lariat, two transesterification reactionstake place: 1) the 2′-OH of the branch point A in the intron to the5′-phosphate of the SD intron G, and 2) the 3′-OH of the SD exon G tothe 5′-phosphate of the first base of the SA exon. The removed intron israpidly degraded in most cases, and the joined exons are now referred toas mature mRNA, which is transported out of the nucleus for translationinto proteins by the ribosome.

B. Targeting Strategies

Various approaches could be taken to targeting the processing (splicing)of mRNA by antisense oligomers. The following sites could be targeted:

-   1. The exon-only portion of the SD exon (upstream of SD site), to    interfere with SD processing.-   2. The SD exon/intron junction, to interfere with SD processing.-   3. The site of lariat formation within the intron, to block    spliceosomal recognition upstream of the SA exon.-   4. The SA intron/exon junction, to interfere with SA processing.-   5. The exon-only portion of the SA exon (downstream of SA site), to    interfere with SA processing.

Prior alt methods have utilized strategy 2 or 4 (targeting of SD or SAjunction). See, for example, RV Giles et al., cited above, in which amorpholino antisense oligomer spanning a splice acceptor site in thec-myc gene is described.

Experiments in support of the present invention found that targetingstrategies 4 and 5 above, directed to the splice acceptor, were morereliably effective than strategies 1 and 2, directed to the splicedonor.

For example, studies were carried out with rat CYP3A2 pre-mRNA targetedin vivo (whole animal). Animals were injected i.p. with 100 μg PMO (asshown in FIG. 2B-B, where Y₁ and Z are oxygen and X is N(CH₃)₂) inphosphate buffered saline. The diminished rate of microsomal metabolismof erythromycin O-demethylase was monitored to reflect the expectedphenotype caused by the antisense inhibition. As shown, the splice donor(SD) targeting was less effective than the splice acceptor (SA)strategy. ANTISENSE SEQUENCE SEQ (/ indicates ID ERDEM % STRATEGY splicejunction) NO: of control Control Saline —  100 ± 10.2 (N = 7) SD3′-AAGAGATGGC/CACTCACTGG-5′ 4 94.7 ± 5.4 (N = 3) SA3′-GGAAATATC/TGAACCTTGGG-5′ 5 86.5 ± 3.8 (N = 3)

Experiments with oligomers antisense to c-myc mRNA were conducted incultured rat NRK cells, evaluating the inhibition of cellular DNAsynthesis by monitoring incorporation of tritated thymidine. Sequenceswere derived from Genbank Acc. No. Y00396 (rat) and J00120 (human),targeting the splice acceptor region at the beginning of exon 2 (withthe exception of SEQ ID NO: 1, which targets the splice donor). The ratand human sequences indicated are highly homologous in this region. Theoligomers listed in Table 1, below, were screened for antiproliferativeeffects using several proliferation assays. Primary fibroblasts from twospecies, rat and human, NRK and WI-38, respectively, were used for thescreening. Data shown in Table 1 employed NPK cells.

Data from [³H]thymidine incorporation assays using 20 μM PMO (as shownin FIG. 2B-B, where Y₁ and Z are oxygen and X is N(CH₃)₂) are presentedin Table 1. Percentages refer to [³H]thymidine incorporation relative tothe vehicle (H₂O) control. Therefore, the lower the number, the greaterthe antiproliferative effect. It can be seen that all oligomers testedexhibited at least some antiproliferative activity. The extent of theinhibitory activity compared favorably with the antiproliferative drugTaxol (Paclitaxel, Bristol-Myers Squibb, Princeton, NJ) at 32% ofcontrol. As 10%-20% of the cells are not affected by the scrape loadingprocedure and will, therefore, contribute to the residual [³H]thymidineincorporation activity, it is likely that most or all cells containingthe most efficacious oligomers were growth inhibited. TABLE 1 CellGrowth Inhibition by Anti-ε-myc and Control Sequences SEQ IncorporationID Targeted vs. vehicle NO: Antisense Sequence (5′→3′) Region* control 1 CTGTGCTTAC/CGGGTTTTCCACCTCCC 2553-2579 51 ± 8% (/ = SD site)  2ATCGTCGTGACTGT/CTGTTGGAGGG 4140-4164 27 ± 3% (/ = SA site)  3GCTCACGTTGAGGGGCATCG 4161-4180 38 ± 2% 25 ACGTTGAGGGGCATCGTCGC J00120  29% 4515-34 42 GGGGCAUCGUCGUGACUGU/CUGUUGGAGGG 4140-4169   20% 43CGUCGUGACUGU/CUGUUGGAGG 4141-4162   45% 44 CGTCGTGACTGT/CTGTTGGAGG4141-4162   21% 45 GGCAUCGUCGCGGGAGGCUG/CUGGAGCG J00120   22% 4498-450546 CCGCGACAUAGGACGGAGAGCAGAGCCC 4364-4391   56% 47 ACTGTGAGGGCGATCGCTGC— ˜100% (scrambled) 48 ACGATGAGTGGCATAGTCGC — >100% (3 mismatches) 49CTCCGCAATGCTGAAAGGTG — >100% (rat BCL-2) 50 GGCGUGCCUCAAACAUGGUGGCGG —˜100% (rat PCNA-1)*Genbank Y00396 (rat) unless otherwise indicated

Two irrelevant-sequence oligomers (rat BCL-2, SEQ ID NO: 49 and ratPCNA-1, SEQ ID NO: 50) did not inhibit NRK cells at the highestconcentration assayed (20 μM). Mismatched and scrambled sequences of SEQID NO: 25 (SEQ ID NOs 48 and 47, respectively) had no effect on theproliferation of WI-38 (human) fibroblasts at 20 μM.

SEQ ID NO: 45, which spans the 3′-splice acceptor site of the firstintron of human myc pre-mRNA, was shown by Giles et al. (1999) to causemissplicing of myc pre-mRNA. A cryptic or latent splice acceptor, 44 bpdistal to the normal splice acceptor position, was used to produce anaberrantly spliced mRNA with a 44-bp deletion. This misspliced mRNAlacks the initiator AUG and did not produce a normal myc protein.

SEQ ID NO: 25 does not span the intron 1-exon 2 boundary, the firstpotential complementary base pair being the tenth nucleotide from the 3′splice acceptor. Similarly, SEQ ID NO: 3 has its 5′ end eleven basesdownstream of the splice acceptor in the rat c-myc mRNA sequence. As thedata shows, both sequences were effective in inhibiting cell growth.

It was of interest to determine if these oligomers inhibited normalsplicing, as had been shown for SEQ ID NO: 45. Accordingly, RNA wasprepared from human cells treated with positive (SEQ ID NO: 45) andnegative (SEQ ID NO: 50) control oligomers as well as SEQ ID NO: 25. Thestructure of the RNA was then analyzed by making a DNA copy with reversetranscriptase and then performing PCR with flanking DNA primers (seeMaterials and Methods, below). The products of the RT-PCR procedure wereanalyzed by agarose gel electrophoresis.

In untreated or irrelevant oligomer-treated cells, a 304-bp band ispredicted, and was observed, from the primers used and the c-mycnucleotide sequence. For cells treated with SEQ ID NO: 25, two DNA bandswere observed. The upper band, which comigrated with the fragment fromuntreated or negative control cells, represented mRNA from the 10%-20%of cells not scrape loaded by the uptake procedure used, plus thefraction of correctly spliced mRNA in treated cells. The heavier lowerband represented the misspliced, 44-bp depleted mRNA.

Cells loaded with SEQ ID NO: 25 also produced two bands from the RT-PCRprocedure, one the size of normally spliced mRNA and one smaller. It cantherefore be concluded that SEQ ID NO: 25, despite not directlyoverlapping the splice acceptor site, is capable of causing missplicing.The irrelevant PMO (SEQ ID NO: 50) gave an mRNA structure patternidentical to that of untreated cells, demonstrating that the missplicingis sequence specific.

A dose-response study with SEQ ID NO: 25 gave an IC₅₀ of 3 μM. Theinhibitory effects began to plateau at 10 μM, and there was littlefurther change from 10 μM to 20 μM.

Myc protein has been implicated as important for the transition fromG₀/G₁ of the cell cycle into the S phase (MK Mateyak et al., Cell GrowthDiffer. 8:1039-48, 1997). It could, therefore, be expected that if mycprotein levels are reduced, the cells would arrest in G₁. This effectwas investigated by determining the number of cells in G₁ and G₂, usingthe method of Telford et al. (Cytometry 13:137-43,1993) (see Materialsand Methods). After removal of RNA by hydrolysis, cells were stained bypropidium iodide, a DNA-specific fluorescent dye. The DNA content percell distribution was then determined by FACS analysis. The FACSintensity profiles show two peaks, corresponding to 2N DNA content (G₁)and 4N DNA (G₂). Cells treated with the PMO having SEQ ID NO: 25 showedan increase in the proportion of cells in G1 (79% compared to 66%vehicle control) compared with those in G₂ (9% compared to 21% vehiclecontrol). A positive control of quiescent cells obtained by growthfactor starvation showed 80% of cells in G₁ and 8% in G₂.

To obtain further evidence that the anti-c-myc oligomer SEQ ID NO: 25inhibits c-myc mRNA expression by an antisense mechanism, a reportergene model system was constructed to directly examine one gene(luciferase) and its activity (light production). The myc-luciferasereporter plasmid contained the entire 2.2-kb 5′ region of human c-mycand the first 6 amino acid codons of the myc protein fused to the insectluciferase cDNA (see Hudziak et al., Antisense Nucleic Acid Drug Dev.10:163-76 (2000). It was transfected into HeLa cells, and aluciferase-producing clone was selected and designated clone L6. Thiscell line was scrape loaded with the indicated concentrations of PMO andreplated. After 24-30 hours, the cells were lysed, and luciferaseactivity was measured (see Materials and Methods).

The results showed strong inhibition of luciferase production, with anIC₅₀ of 300 nM. Several control experiments were performed to determineif the observed inhibition of luciferase synthesis was due tosequence-specific inhibition by the PMO. SEQ ID NO: 25 and the twosequence permutations noted above (SEQ ID NOs: 47 and 48) were compared.Neither the scrambled version nor the 3-base mispair oligomer had anyeffect on luciferase production. Other control experiments showed thatSEQ ID NO: 25 had no effect on HeLa proliferation during the 30-hourincubation time of the luciferase assay, and had no effect on anunrelated target reporter system (rabbit α-globin; J Summerton et al.,Antisense & Nucleic Acid Drug Dev. 7:63-70, 1997) under conditions wherea sequence complementary agent gave 70% inhibition. Other sequencescontaining 3-4 contiguous guanine (G) bases were tested in the NRK cellproliferation assay described above, and gave no significant inhibitionof cell proliferation at concentrations up to 20 μM.

In an evaluation of the “functional footprint” of PMO antisense directedto the splice acceptor (SA) region of rat c-myc, interference insplicing was observed with PMOs targeting the region from −44 upstreamof the SA to +36 downstream (3′ end of target region) from the SA. Asthe target sequence moved downstream, the proportion of lower molecularweight proteins, relative to normal c-myc, was observed to increase.

Targeting downstream of the splice acceptor junction, i.e. within theexon, is generally preferred, for reasons discussed below.

IV. Consequences of Interference with SA Site

If a binding oligomer interferes with normal mRNA splicing at the SAsite, the spliceosome will proceed to the next best unblocked candidatesite in the region. This site tends to be a [C]AG sequence with a shortrun of pyrimidines 10 to 15 bases upstream and a suitable branch pointfurther upstream.

The resulting processed mRNA will generally be a splice variant mRNA inwhich the sequence between the normal splice acceptor site and thealternate (or “cryptic”) splice acceptor site has been deleted. Theresulting variant protein can take different forms depending on thedeleted sequence. For example, if the deleted sequence contains a wholemultiple of three base pairs (that is, the downstream splice acceptorsite is a whole multiple of three bases downstream of the normal spliceacceptor site), the subsequent sequence will be in frame with the normalsequence, and a truncated form of the native protein will result. Thispermits the formation of dominant negative proteins, as described, forexample, in Example C below. If the cryptic site is out of frame withthe normal site, however, an unrelated “nonsense” protein will beproduced. Due to the frequent occurrence of stop codons in non-readingframe sequences (about one per twenty codons), such a splice variantmRNA typically results in early termination of translation.

If the deleted sequence includes an AUG start site, translation may thenoccur at an alternate AUG site found further downstream. Again, if thisalternate site is in frame with the normal AUG site, the resultingprotein will have a deletion of some number of amino acids from thenative protein. If the alternate site is out of frame, a “nonsense”protein, typically truncated early in translation, will result.

In one embodiment, exemplified in Examples A and B below, acarboxy-terminal truncated protein is created. In another embodiment,exemplified in Examples C and D below, a protein having an abnormal ordeleted amino terminus is produced. The latter can be accomplished ifthe AUG translation start site is in exon 2 or greater of the mRNA.

Thus, when the sequence of pre-mRNA for a desired protein is known, anantisense target can be designed to alter desired regions of theprotein. Preferably, the binding domains of the protein are also known,such that selected functions of the protein can be altered.

The following steps can be used to target genes with multiple exons, inaccordance with the invention:

-   Step 1. Identify functional domains of the protein in question. The    scientific literature can provide most of this information. One    excellent source is “The Oncogene FactsBook” by Robin Hesketh,    Academic Press, London, 1995, in which proteins related to cancer    are reviewed and their functional domains mapped. Similarly, the    “Cytokine FactsBook” (R E Callard and A J H Gearing, Academic Press)    describes functional domains of cytokines. Other works in the same    series include “The Protein Kinase Factsbook”, “The G-Protein Linked    Receptor Factsbook”, and “The Extracellular Matrix Factsbook”.-   Step 2. Search GenBank or similar nucleic acid databases for the    gene sequence, including exon/intron sequences and junctions.-   Step 3. Preferred antisense targets are within approximately 35-40    downstream bases of a splice acceptor site. Preferably, the target    region has its 5′ end at a location 1 to 25 bases downstream, more    preferably 2 to 20 bases downstream, and most preferably 2 to 15    bases downstream, from the SA junction. (Note that a sequence one    base downstream directly abuts, but does not overlap, the [C]AG    splice acceptor site, and includes the first base of the normal    exon.)-   Step 4. To predict the consequence of inhibition, search the exon    downstream of the SA for a cryptic splice acceptor, i.e. a [C]AG    sequence downstream from the authentic SA. Once such a candidate    site is located, search for a 5′-TACTAAC-3′ (or similar) site for    lariat formation 12 to 50 bases upstream of the cryptic SA. (Note    that, according to the consensus sequence given above, only the    branch point A must be conserved, so there is considerable    flexibility in this sequence.) Preferably, there should also be a    short pyrimidine-rich region 10-15 bases upstream of the cryptic SA.-   Step 5. Once a potential cryptic SA site is identified, determine if    its use will result in “in frame reading” by dividing the number of    bases between the authentic site and the cryptic site by 3. If the    quotient is a whole number, then the resultant protein will be “in    frame” and possibly a dominant negative protein (see below).

Accordingly, in one preferred embodiment, an antisense target is chosensuch that splicing is likely to be directed to a cryptic site whose usewill result in in-frame reading.

V. Target Proteins and Selected Examples

Suitable target proteins include, for example, transcription factors,particularly oncogenic or proto-oncogenic proteins such as myc, myb,rel, fos, jun, abl, bcl, and p53; matrix proteins, such as integrins andcathedrins, other tumor-expressed proteins, such as hCG; telomerases;receptor proteins, cytokines; kinases, and viral proteins, such as HIVrev, human papilloma virus, and human parvovirus B19. It is appreciatedthat inhibition of such proteins has numerous therapeutic applications.These include, but are not limited to, antitumor therapy, e.g. bytargeting proteins, such as transcription factors, involved in variousaspects of cell division and cell cycle regulation; antiviral orantibacterial therapy, by targeting proteins essential to replication orother vital functions of the infectious agent; and inhibition ofrestenosis or other proliferative disorders, by inhibiting proteinswhich support cell proliferation at the site.

Transcription factors are typically multidomain proteins, having a DNAbinding region and a protein-protein binding region. Interfering withone of these regions can produce a dominant negative protein, whichcounters the activity of the native protein by preserving one activity(such as protein binding) while inhibiting another activity critical tothe proper function of the protein (such as DNA binding; or vice versa).See the c-myc example described below.

As noted above, functional domains of many of the target proteins notedabove have been studied extensively and reported in the literature.Sequences of pre-mRNA, including locations of introns, exons, and AUGstart codons, can be found in the GenBank sequence database or otherpublished sources readily available to those of skill in the art.

Following are several examples of antisense targeting downstream ofsplice acceptor domains in selected proteins to produce splice variantsmRNAs which, upon translation, produce proteins with specificalterations.

In one embodiment, exemplified in Examples A and B, a carboxy-terminaltruncated protein is created. In another embodiment, exemplified inExamples C and D, a protein having an abnormal or deleted amino terminusis produced.

A. Human Androgen Receptor (GenBank M35845, M35846)

A review of prostate cancer molecular biology indicates that androgenablation is the state of the art therapy. In accordance with the presentinvention, this can be accomplished by inactivation of the androgenreceptor. By targeting the center of the gene, at exon 2, variousoptions are available for inhibiting expression of a functional androgenreceptor. Proposed targeting of the splice acceptor in exon 2 (SEQ IDNO: 8; GenBank M35845) or exon 3 (SEQ ID NO: 11; GenBank M35846) isindicated. End of Intron 1: (SEQ ID NO: 6) 5′-...TGTGTCTTTTCCAG/

 splice acceptor site Exon 2: (SEQ ID NO: 7)5′-TTTGGAGACTGCCAGGGACCATG...-3′ Target antisense sequence: (SEQ ID NO:8) 5′-CATGGTCCCTGGCAGTCTCC-3′

This oligomer (SEQ ID NO: 8) targets the sequence starting at base 48,four bases downstream of the normal splice acceptor site. The nextprobable cryptic splice site is at base 100 (CAG at 97-99; branch pointA at 80; upstream pyrimidine region at 87-92. This site is out of framewith the normal site and should thus result in early termination of theprotein.

Similar length oligomers targeting sequences having a 5′ end 1 to about18 bases downstream of the splice acceptor could also be used. Thisincludes oligomers having a length of about 18 contiguous nucleotidesselected from the sequence 5′-TCA ATG GGC AAA ACA TGG TCC CTG GCA GTCTCC AAA-3′ (SEQ ID NO: 9, complementary to bases 45-80 of the sequencegiven in Genbank Acc. No. M35845). End of intron 2: (SEQ ID NO: 10)5′-...TTTGTGTTCTCCCAG/

 splice acceptor site Exon 3: (SEQ ID NO: 11)5′-GGAAACAGAAGTACCTGTGCGCC...-3′ Antisense sequence: (SEQ ID NO: 12)5′-GGC GCA CAG GTA CTT CTG-3′

This oligomer (SEQ ID NO: 12) targets the sequence starting at base 49,six bases downstream of the normal splice acceptor site. A possiblecryptic splice site is at base 145 (CAG at 143-145, branch point A at114; pyrimidine region at 123-127). This site is in frame with thenormal splice site and should thus result in a carboxy-truncated versionof the native protein.

Similar length oligomers targeting sequences having a 5′ end 1 to about18 bases downstream of the splice acceptor could also be used. Thisincludes, for example, oligomers having a length of about 18-20contiguous nucleotides selected from the sequence 5′-AAT CAT TTC TGC TGGCGC ACA GGT ACT TCT GTT TCC-3′ (SEQ ID NO: 13; complementary to bases44-79 of the sequence given in Genbank Acc. No. M35846).

B. Human chorionic gonadotropin (β subunit) (GenBank X00266)

The β subunit of hCG is nearly identical to leutinizing hormone (LH)with the exception of the COOH end, which is extended in hCG. Thisextension is in exon 3 of the gene. By interfering with the exon 3 SAsite, hCG could be truncated without targeting LH, unlike targeting ofthe AUG translation initiation sites, which are highly conserved in bothproteins.

An oligomer with the sequence 5′-CCC CTG CAG CAC GCG GGT-3′ (SEQ ID NO:14) binds in exon 3 near (directly abutting) the SA (CAG at 1318-1320),targeting the sequence from bases 1321-1338, and interferes withsplicing at this site. Similar length oligomers targeting sequenceshaving a 5′ end at base 1322, 1323, etc., up to about base 1340, couldalso be used. This includes, for example, oligomers having a length ofabout 18-20 contiguous nucleotides selected from the sequence 5′- GAGGCA GGG CCG GCA GGA CCC CCT GCA GCA CGC GGG T-3′ (SEQ ID NO: 15;complementary to bases 1321-57 of the sequence given in Genbank Acc. No.X00266).

Possible cryptic SA sites are at base 1393 (AG at 1391-2; branch point Aat 1370 or 1373; in frame) and at 1458 (CAG at 1455-7; branch point A at1427; out of frame). Splicing at either site would delete at least 24amino acids from the protein, and possibly more, if the out of framesite were used. Since hCG is expressed only in tumor cells, this wouldbe therapeutically beneficial in that less protein is expressed, and theprotein will have a shorter biological half-life. In addition, thetruncated protein may have unusual amino acids at the COOH end, possiblyproducing an immune response to hCG, useful in a vaccination strategy.

C. Human c-myc (GenBank J00120)

c-myc is a proto-oncogene which regulates cell growth anddifferentiation and is involved in the processes of vascular remodeling,smooth muscle cell proliferation, extracellular matrix synthesis, andapoptosis. Aberrant expression of c-myc is frequently observed in humancancer. Aberrant, constitutive or overexpression of c-myc has beenassociated with a number of human cancers including lung cancer,colorectal cancer, breast cancer, bladder cancer, leukemia, lung cancer,etc. It has also been demonstrated that inhibition of c-myc reduces theincidence and severity of restenosis.

The c-myc protein has a DNA binding domain in the amine-terminal portionof the sequence and a protein-protein interacting domain in thecarboxy-terminal portion. It is known that c-myc binds with Max in thecarboxy domain to form a heterodimer that can bind to a DNA sequenceknown as an E-Box (5′-CACGTG-3′). When myc:max binds in this manner, thephenotype is growth stimulatory and can lead to apoptosis.

If myc concentration is low, then max foil homodimers which do nottransactivate. If the mad protein is induced, then max binds mad to formmad: max heterodimers, which tend to induce differentiation and areanti-apoptotic.

Hence, simply inhibiting myc tends to result in the mad:max phenotype,which is anti-apoptotic. However, if the DNA binding (amine-terminal)domain of c-myc is inhibited, while leaving the protein-protein binding(carboxy-terminal) domain intact, the result is a dominant negativeprotein capable of binding max but not capable of transactivation. Thefavorable phenotype would be the loss of growth stimulatory actions, butthe compensatory anti-apoptotic actions of mad:max would not dominate,as max protein can still bind to the myc COOH domain.

Inhibition of the amine-terminal domain is achieved by appropriatedesign of an antisense compound to direct alteration of splicing.Cryptic (alternate) SA sites found downstream include:

-   4547 Possible candidate; the next AUG, at 4554, is out of frame and    would thus produce unrelated (“nonsense”) protein. (Could be blocked    by oligos targeted further downstream than SEQ ID NO: 13)-   4578 Not a good SA candidate, due to few pyrimidines upstream-   4617 A good SA candidate; next downstream AUG is at 4821, which is    in frame with normal AUG at 4521

As described above, a morpholino oligomer (PMO) having a sequencecomplementary to bases 4515 to 4534 of the human c-myc mRNA sequence(Genbank Accession No. J00120), ten bases downstream of the spliceacceptor site AG at 4504-5 (SEQ ID NO. 25, 5′-ACG TTG AGG GGC ATC GTCGC-3′), was found to prevent appropriate splicing at this site and theuse of the normal AUG translation start site at 4521. Analysis of themRNA produced indicated use of the 4617 site indicated above.Translation starting at the AUG at 4821, 300 bases downstream of thenormal AUG, produces a protein having a 100 amino acid deletion at theN-terminus (as also reported by Giles et al.) Use of antibodies to theCOOH end of myc revealed this protein, as well as substantially smallerproteins, from cells treated with the antisense oligomer. TheseN-terminal truncated proteins are expected to bind max but will not bindDNA.

These results, combined with examination of primer-amplified mRNAs,demonstrated the use of the 4617 cryptic splice site, as well as othercryptic sites further downstream.

As noted above, PMO oligomers targeted to the region from −44 upstreamof the SA to +36 downstream (ds) from the SA showed evidence ofinterference with splicing in rat c-myc. Assuming an oligomer length ofabout 20 bases, the following antisense sequences could thus be utilizedfor targeting of human c-myc: TABLE 2 SEQ Bases ds of SA site IDAntisense Sequence (5′end of sequence) NO: 5′-GGCATCGTCGCGGGAGGCTG-3′ 116 5′-GGGCATCGTCGCGGGAGGCT-3′ 2 17 5′-GGGGCATCGTCGCGGGAGGC-3′ 3 185′-AGGGGCATCGTCGCGGGAGG-3′ 4 19 5′-GAGGGGCATCGTCGCGGGAG-3′ 5 205′-TGAGGGGCATCGTCGCGGGA-3′ 6 21 5′-TTGAGGGGCATCGTCGCGGG-3′ 7 225′-GTTGAGGGGCATCGTCGCGG-3′ 8 23 5′-CGTTGAGGGGCATCGTCGCG-3′ 9 245′-ACGTTGAGGGGCATCGTCGC-3′ 10 25 5′-AACGTTGAGGGGCATCGTCG-3′ 11 265′-TAACGTTGAGGGGCATCGTC-3′ 12 27 5′-CTAACGTTGAGGGGCATCGT-3′ 13 285′-GCTAACGTTGAGGGGCATCG-3′ 14 29 5′-AGCTAACGTTGAGGGGCATC-3′ 15 305′-AAGCTAACGTTGAGGGGCAT-3′ 16 31 5′-GAAGCTAACGTTGAGGGGCA-3′ 17 32

The protein-protein binding (carboxy terminal) domain of myc could alsobe altered, as follows. An oligomer with the antisense sequence 5′-TCCTCA TCT TCT TGT TCC TC-3′ (SEQ ID NO: 33) targets base 6656, downstreamof the splice acceptor at base 6654-5. Likely downstream cryptic SAsites are at bases 6704, 671 0, and 6729 (AG at 6702-3; CAG at 6707-09;CAG at 6726-8; A branch point at 6684; pyrimidine-rich region startingat 6690). Of these, the first two are out of frame and the third is inframe. Use of the third site would be expected to produce a deletion of75 base pairs from the mRNA, resulting in a 25 amino acid deletion inthe protein-protein binding domain of the myc protein. This proteinwould be the converse dominant negative, as DNA binding may be possiblebut no myc:max transactivation is likely. The function of myc would belost, but the mad:max heterodimers would be favored, so that thephenotype of differentiation and anti-apoptosis would be observed.

As noted above, oligomers targeted progressively farther downstream,relative to SEQ ID NO: 33, could also be used. This includes, forexample, oligomers having a length of about 18-20 contiguous nucleotidesselected from the sequence 5′-AAC AAC ATC GAT TTC TTC CTC ATC TTC TTGTTC CTC-3′ (SEQ ID NO: 34, complementary to bases 6656-91 of thesequence given in Genbank Acc. No. J00120). In accordance with theinvention, an oligomer targeted far enough downstream to inhibitsplicing at the first two cryptic sites noted above could be effectiveto promote splicing at the third cryptic site.

D. Human p53 (GenBank X54156)

Like c-myc, p53 has a non-coding exon 1, a large intron 1 and an AUGstart codon near the SA site of exon 2. An oligomer targeted, forexample, to the region having its 5′ end at base 11691, three basesdownstream of the SA site (5′-CCC GGA AGG CAG TCT GGC-3′; SEQ ID NO: 35)is expected to interfere with translation at the AUG initiation site aswell as the normal splicing of exon 2. As described for c-myc above,other suitable oligomers include those targeted one or two basesdownstream of the SA site, or targeted progressively further downstream,e.g. starting at base 11691, 11692, etc., and targeting some portion ofthe region between base 11689 (the first base of the normal exon 2) andabout base 11725. This includes, for example, oligomers having a lengthof about 18-20 contiguous nucleotides selected from the sequence 5′- TCCTCC ATG GCA GTG ACC CGG AAG GCA GTC TGG CTG -3′ (SEQ ID NO: 36;complementary to bases 11689-11724 of the sequence given in Genbank Acc.No. X54156).

Cryptic SA sites are available at base 11761 (AG at 11759-60) and atbase 11765 (CAG at 11762-4) (A branch point at 11736; pyrimidine run at11750-57). The next AUG, at base 11782, is out of frame, which willresult in nonsense proteins. Alternatively, initiation may begin in exon3, producing truncated p53-type proteins without the p53 amino terminus.

The following is an example in which targeting of the splice acceptor isespecially advantageous.

E. Human Abl (GenBank AJ131466)

Since the bcr gene breaks and fuses to abl, forming the bcr-abl fusionprotein, in chronic myeloid leukemia, it is a target of antisenseinhibition. With respect to abl, fusion can occur at various locations;i.e. there are breaks after exon 1, 2 and 3 of bcr that fuse to Exon 2of abl. However, by targeting the splice acceptor of abl, only oneoligomer is required for treatment of all CML patients.

The bcr-abl fusion point is at the junction of bases 373-374. Therefore,the abl splice acceptor could be targeted by the following sequence,three bases downstream of this junction: 5′-CTA CTG GCC GCT GAA GGG C-3′(SEQ ID NO: 37).

Again, other oligomers targeting the region between the splice junctionand about 35-40 bases downstream of the splice junction could also beused. This includes, for example, oligomers having a length of about18-20 contiguous nucleotides selected from the sequence 5′- GCT CAA AGTCAG ATG CTA CTG GCC GCT GAA GGG CTT -3′ (SEQ ID NO: 38; complementary tobases 374-409 of the sequence given in Genbank Acc. No. AJ131466).Possible cryptic splice sites (both out of frame) include CAG motifs at468-70, with an upstream pyrimidine region at 453-459 and an A branchpoint at 421, and at 516-518, with an upstream pyrimidine region at507-510 and an A branch point at 485.

F. HIV-1 (GenBank L39106)

This example illustrates a situation in which the method of theinvention provides particular advantages; that is, where both a targetvirus and the host (human) express a gene with the same mRNA sequences.The protein products are important to the function of both virus andhost but serve different functions.

An mRNA sequence of HIV-1 rev, which encodes a protein critical forviral replication (see e.g. H Mitsuya et al., Science 249:1533-1543,1990), was found to also occur in the host, leading to toxic effectswhen this sequence was inhibited by antisense. Specifically, aphosphorothioate oligomer having the sequence 5′-TCG TCG GTC TCT CCG CTTCTT CTT GCC-3′ (SEQ ID NO: 39) was used to inhibit HIV-1 rev (Matsukuraet al., PNAS USA 86:4244-4248, 1989). This region of rev is highlyconserved in HIV; accordingly, it was targeted so that a large varietyof viral isolates might be inhibited.

However, the preclinical development of this 27-mer was ended when 2 of3 Rhesus monkeys treated by continuous infusion died of what appeared tobe opportunistic infections. An excessive endotoxin burden wassuspected, so the experiment was repeated, with care taken to removeendotoxin. However, two cynamologous monkeys also died on days 8 and 9of the continuous infusion in the repeat study. As endotoxin waseffectively removed from consideration, immunosuppression was suggestedas the cause. The white blood cell (WBC) count in the three Rhesusmonkeys was 9.5+0.7 prior to infusion with the antisensephosphorothioate to HIV-rev, and fell to 6.9+0.6 during the infusion.There was no associated change in RBC or hematocrit. Further, surfacemarker studies for cells involved in immune response were influenced:CD2 was reduced from 88 to 76, CD8 fell from 45 to 36, and CD20 rosefrom 14 to 18.

A homologous region to HIV-rev observed in the genomes of humans andmonkeys was reported in J. Virology 66:2170-2179 (1992), thus accountingfor these toxic effects.

According to the present method, an alternative sequence could beselected which is less likely to interfere with host processes. Inaccordance with the present invention, HIV-rev could be suppressed asfollows.

The protein is encoded by two exons: exon 1, 5493 . . . 5568, 76 bases;and exon 2, 7885 . . . 8180, 296 bases; 124 amino acids total (GenBankL39106). An antisense, RNAse H-incompetent oligomer targeted to theregion between the splice junction (i.e. the first base of normal exon2) and about 35-40 bases downstream of the splice junction is expectedto interfere with splicing of the pre-mRNA. As specified above, the 5′end of the targeted region is preferably 1 to about 25 bases downstreamof the splice junction. An example of such an oligomer is a PMO targetedto base pairs 7885-7904, having the sequence 5′-CTC TGG TGG TGG GTA AGGGT-3′ (SEQ ID NO: 40). Other candidates include oligomers having alength of about 18-20 contiguous nucleotides selected from the sequence5′-CGG GTC TGT CGG GTT CCC TCT GGT GGT GGG TAA GGG T-3′ (SEQ ID NO: 41;complementary to bases 7885-7921 of the sequence given in Genbank Acc.No. L39106).

The most likely site for a cryptic splice acceptor is at base 7975,where an AG sequence is preceded by multiple pyrimidines over 10 basesupstream from the AG. If this cryptic splice-acceptor is utilized, adeletion of 90 bases, or 30 amino acids, will result. This deletion willinterfere with effective viral rev gene function.

VI. Analysis of Effects of Splice Acceptor Targeting

The effectiveness of a particular antisense sequence in producing splicevariant mRNA may be determined by known analytical methods. For example,the presence or absence of the encoded, full-length protein and oftruncated or other variant proteins can be monitored by standardtechniques such as ELISA or Western blotting. Antibodies targeted tospecific regions of the proteins, e.g. to the carboxy or amino terminus,can also be employed.

mRNA structure can be analyzed to evaluate antisense oligomer inducedmissplicing. The recovery of nuclear RNA is essential to observeintron-containing hnRNA, as the nucleus is the site of intron removal.Preparation of nuclear RNA is described in books such as “MolecularCloning, A Laboratory Manual” (T. Maniatis, E. F. Fritsch and J.Sambrook, eds., Cold Spring Harbor Press) or “Current Protocols inMolecular Biology” (F. M. Ausubel et al., eds., John Wiley & Sons,Inc.). The analysis of hnRNA is best done by either Northern blot or S1Mapping. The presence of particular splice variant mature mRNAs can bedetermined by carrying out PCR amplification using selected primer pairshaving sequences predicted to occur in a specific splice variant (ornormal) processed mRNA. One primer is positioned in the SD (splicedonor) exon upstream of the splice site, and the second is positioned inthe SA (splice acceptor) exon, downstream of the splice site. Since theoligomer interferes with the SA site, the downstream primer should bemore than 50 bases downstream from the SA splice site. Analysis of thePCR reactions on agarose gels stained with ethidium bromide will revealamplified bands which are smaller in size than bands observed fromuntreated control cells, as evidence of blocking of the SA site by theoligomer.

VII. Advantages of the Method

The present invention demonstrates that inhibition or alteration ofprotein expression can be achieved by antisense targeting of a regiondownstream of a splice acceptor, in the coding region of a pre-mRNA,using a non-RNAse competent oligomer. The present compositions andmethods have several advantages over prior art methods of antisenseinhibition, in which the targeting antisense compound spans andhybridizes to a splice acceptor sequence (e.g. Giles, cited above; Koleand Dominski, U.S. Pat. No. 5,665,593).

One such advantage is that exon sequences tend to much more highlyconserved among species than intron sequences. This allows for greaterpredictability in testing such methods on animal models.

In addition, greater flexibility is allowed in selecting a sequence fortargeting, as the oligomer is not required to actually bind to a precisesite such as a splice acceptor junction or an AUG start codon. Thepresent invention thus increases the range of sequences which may besuccessfully targeted in an antisense application. Such flexibility canbe advantageous in avoiding undesirable side reactions, such as causedby inadvertent targeting of non-target proteins in a subject, ortargeting of host proteins when attacking an infectious agent such as avirus or bacterium. This is demonstrated in Example F, above.

In the present strategy, in contrast to methods which target the splicejunction directly, various regions of the exon downstream of the SA sitemay be blocked. This opens the possibility of producing differentvariant proteins, by directing splicing to different cryptic splicesites downstream of the normal SA. As described above, certain variantproteins, such as dominant negative proteins, can have uniqueadvantages. Accordingly, the antisense could be designed to promotesplicing at a particular cryptic splice site, which would give rise tothe desired variant protein, over others. For example, a less desirablecryptic splice site close to the normal SA site could be hindered infavor of a site further downstream.

Further benefits of flexibility of design may include convenience ofsynthesis or enhanced binding affinity. In addition, by using thepresent strategy of targeting fully within the exon, the target RNA canbe identified by the antisense oligomer in either the nucleus or thecytoplasm, which can be advantageous for purposes of analysis.

VIII. Treatment Methods

In a related aspect, the invention includes a method of treating orpreventing a disease state by inhibiting or altering expression of atarget protein. Such disease states include viral, bacterial or fungalinfections, cancerous tumors, and other conditions characterized bycellular proliferation, such as restenosis, hyperproliferative skindisorders, or inflammation. Proteins targeted, as noted above, includetranscription factors, which include many oncogenes, receptor proteins,matrix proteins, and viral proteins. Inhibition of such proteinsgenerally results in disruption of the cell cycle, viral replication, orother critical functions.

The method is carried out by administering to the subject an antisenseoligomer 12 to 25 nucleotides in length and having (i) a base sequencecomplementary to a target region of a selected preprocessed mRNA codingfor the target protein, where the 5′ end of the target region is 1-25bases downstream, and preferably 2-15 bases downstream, of a normalsplice acceptor site in the preprocessed mRNA, and (ii) a substantiallyuncharged morpholino backbone, preferably containing aphosphorodiamidate backbone as shown in FIG. 2B-B, where X═NH₂, NHR, orNR₂ (where R is lower alkyl, preferably methyl), Y═O, and Z=O. Alsopreferred are morpholino oligomers where the phosphordiamidate linkagesare a mixture of uncharged linkages as shown in FIG. 2F and cationiclinkages as shown in FIG. 2G where, in FIG. 2B-B, X=1-piperazino.

In general, the method comprises administering to a subject, in asuitable pharmaceutical carrier, an amount of the antisense agenteffective to interfere with splicing at the normal splice acceptor site,and thus suppress normal expression of the protein. In a preferredembodiment, the method results in expression of a dominant negativevariant of the protein. In one aspect of the method, the subject is ahuman subject.

Effective delivery of the antisense oligomer to the target mRNA is animportant aspect of the method. PMOs have been shown to enter cellsefficiently (see e.g. J Summerton et al., Antisense Nucleic Acid DrugDev. 7:63-70, 1997, and copending and co-owned U.S. provisionalapplication 60/117,846). For use in antiviral treatment, varioussystemic routes of delivery, including oral and parenteral routes, e.g.,intravenous, subcutaneous, intraperitoneal, and intramuscular, as wellas inhalation, transdermal and topical delivery, can be used.

Typically, one or more doses of antisense oligomer are administered,generally at regular intervals for a period of about one to two weeks.Preferred doses for oral administration are from about 1 mgoligomer/patient to about 25 mg oligomer/patient (based on a weight of70 kg). In some cases, doses of greater than 25 mg oligomer/patient maybe necessary. For IV administration, the preferred doses are from about0.5 mg oligomer/patient to about 10 mg oligomer/patient (based on anadult weight of 70 kg). Dosages will vary in accordance with suchfactors as the age, health, sex, size and weight of the patient, theroute of administration, and the efficacy of the oligonucleotide agentwith respect to the particular disease state. For treatment ofinfectious agents, a preferred dosage is typically that which isnecessary to attain a concentration in the blood of from about 0.01 toabout 1 μM, and more preferably about 200-400 nM antisense oligomer.This concentration can be achieved in a variety of ways; doses ofbetween about 0.05 and about 0.2 mg/kg/hour by continuous IV infusionhave been found to be acceptable. Greater or lesser amounts ofoligonucleotide may be administered as required.

For treatment of hyperproliferative skin disorders, topicaladministration is indicated. In treatment of restenosis, delivery of theantisense oligomer to the affected cells, that is, to the site ofarterial injury, is recommended. Delivery methods known in the field,such as those described in co-owned and copending U.S. application Ser.No. 09/493,427, can be used to deliver the oligomer to the site ofangioplasty in a patient. Preferably, the oligomer is deliveredconcurrent with the angioplasty procedure. For an adult human, arecommended dosage is in the range of 1-25 μmol of antisense oligomer,and preferably 2-15 mol. With respect to the surface area of tissue tobe treated, an effective dose is typically in the range of 30 to 3000 μgoligomer per cm² of vessel wall, and more preferably about 300 to 1500μg/cm². The patient may also be given the composition on a periodicbasis after angioplasty, at a dosage level sufficient to further inhibitrestenosis.

An effective in vivo treatment regimen using the antisenseoligonucleotides of the invention will vary according to the frequencyand route of administration, as well as the condition of the subjectunder treatment. Optimum dosages for a given route can be determined byroutine experimentation according to methods known in the art. Such invivo therapy is generally monitored by tests appropriate to theparticular type of ailment being treated, and a corresponding adjustmentin the dose or treatment regimen can be made in order to achieve anoptimal therapeutic outcome.

Entry of a morpholino oligomer into cells and binding to its target RNAsequence can be verified by techniques set forth in copending andco-owned U.S. provisional application 60/117,846, which is incorporatedherein by reference. A morpholino antisense compound of the typedisclosed herein, when administered in vivo, can be detected in theurine of the receiving subject in a heteroduplex form consisting of theantisense compound and its RNA complement. This verifies that theantisense compound has been taken up by the target tissue and allows thepractitioner to monitor the effectiveness of the treatment method, e.g.the effectiveness of various modes of administration, and dosages givingmaximal or near-maximal levels of heteroduplex in the urine.

IX. Formulations

A morpholino antisense oligonucleotide composition may be administeredin any convenient physiologically acceptable vehicle. Examples ofstandard pharmaceutically accepted carriers include saline, phosphatebuffered saline (PBS), water, aqueous ethanol, emulsions such asoil/water emulsions, triglyceride emulsions, wetting agents, tablets andcapsules. It will be understood that the choice of suitablephysiologically acceptable carrier will vary dependent upon the chosenmode of administration.

In general, in addition to the active compounds, the pharmaceuticalcompositions of the invention may contain suitable excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Suitable excipientsinclude fillers such as sugars, for example, lactose, sucrose, mannitolor sorbitol, cellulose preparations, calcium phosphates, and binderssuch as starch, gelatin, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinylpyrolidone. If desired, disintegrating agents may be added, such as theabove-mentioned starches as well as carboxymethyl starch, cross-linkedpolyvinyl pyrolidone, agar, alginic acid, or a salt thereof. Auxiliariesinclude flow-regulating agents and lubricants, for example, silica,talc, stearic acid or salts thereof, and/or polyethylene glycol.

For oral administration, dragee cores may be provided with suitablecoatings which are resistant to gastric juices. In order to producecoatings resistant to gastric juices, solutions of suitable cellulosepreparations, such as acetylcellulose phthalate orhydroxypropylmethyl-cellulose phthalate, are used. Concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinylpyrrolidone, polyethyleneglycol, and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.

Other pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer such as glycerol or sorbitol. The push-fitcapsules can contain the active compounds in the form of granules whichmay be mixed with fillers such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds are preferablydissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycols. In addition, stabilizers maybe added.

Pharmaceutical preparations which can be used rectally include, forexample, suppositories, which consist of a combination of the activecompounds with a suppository base. Suitable suppository bases includenatural or synthetic triglycerides, paraffin hydrocarbons, polyethyleneglycols, or higher alkanols. In addition, it is possible to use gelatinrectal capsules which consist of a combination of the active compoundswith a base. Possible base materials include liquid triglycerides,polyethylene glycols, or paraffin hydrocarbons.

Suitable liquid formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble orwater-dispersible form. In addition, suspensions of the active compoundsas appropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension, such as sodiumcarboxymethyl cellulose, sorbitol, and/or dextran. The suspension mayalso contain stabilizers.

In addition to administration with conventional carriers, the activeingredients may be administered by a variety of specialized deliverytechniques. For example, the compounds of the present invention may beadministered encapsulated in liposomes. (See, e.g., Williams, S. A.,Leukemia 10(12): 1980-1989, 1996; Lappalainen et al., Antiviral Res.23:119, 1994; Uhlmann et al., “Antisense Oligonucleotides: A NewTherapeutic Principle”, in Chemical Reviews, Volume 90, No. 4, pp544-584, 1990; Gregoriadis, G., Chapter 14, “Liposomes”, in DrugCarriers in Biology and Medicine, pp 287-341, Academic Press, 1979.) Theactive ingredient, depending upon its solubility, may be present both inthe aqueous phase and in the lipidic layer(s), or in what is generallytermed a liposomic suspension. The lipidic layer generally comprisesphospholipids, such as lecithin or sphingomyelin, steroids such ascholesterol, ionic surfactants such as diacetylphosphate, stearylamine,or phosphatidic acid, and/or other hydrophobic materials. The diametersof the liposomes generally range from about 15 nm to about 5 microns.

Hydrogels may also be used as vehicles for antisense oligomeradministration, for example, as described in WO 93/01286. Alternatively,the oligonucleotides may be administered in microspheres ormicroparticles. (See, e.g., Wu G Y and Wu C H, J. Biol. Chem.262:4429-4432, 1987.) Such vehicles are particularly suited for topicaladministration or in treating restenosis.

Transdermal delivery of antisense oligomers may be accomplished by useof a pharmaceutically acceptable carrier adapted for topicaladministration. One example of morpholino oligomer delivery is describedin PCT patent application WO 97/40854. Other sustained releasecompositions are also contemplated within the scope of this application.These may include semipermeable polymeric matrices in the form of shapedarticles such as films or microcapsules.

Methods for preparing such dosage forms are known or will be apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences (19th Ed., Williams & Wilkins, 1995). The pharmaceuticalpreparations are manufactured according to procedures well known in theart. For example, they may be made by means of conventional mixing,granulating, dragee-making, dissolving, or lyophilizing processes. Theprocess to be used will depend ultimately on the physical properties ofthe active ingredient used.

X. Preparation of Morpholino Oligomers having Cationic Linkages

A schematic of a synthetic pathway that can be used to make morpholinosubunits containing a (1-piperazino) phosphinylideneoxy linkage is shownin FIG. 3. As shown in the Figure, reaction of piperazine and tritylchloride gave trityl piperazine (1a), which was isolated as thesuccinate salt. Reaction with ethyl trifluoroacetate (1b) in thepresence of a weak base (such as diisopropylethylamine or DIEA) provided1-trifluoroacetyl-4-trityl piperazine (2), which was immediately reactedwith HCl to provide the salt (3) in good yield. Introduction of thedichlorophosphoryl moiety was performed with phosphorus oxychloride intoluene.

The acid chloride (4) is reacted with morpholino subunits (moN), whichmay be prepared as described in U.S. Pat. No. 5,185,444 or in Summertonand Weller, 1997 (cited above), to provide the activated subunits(5,6,7). Suitable protecting groups are used for the nucleoside bases,where necessary; for example, benzoyl for adenine and cytosine,isobutyryl for guanine, and pivaloylmethyl for inosine. The subunitscontaining the (1-piperazino) phosphinylideneoxy linkage can beincorporated into the existing PMO synthesis protocol, as described, forexample in Summerton and Weller (Anisense Nucleic Acid Drug Dev.7:187-95, 1997), without modification.

Materials and Methods

Oligomers

PMO's were synthesized at AVI BioPharma by methods described, forexample, in Summerton and Weller, Antisense & Nucleic Acid Drug Dev.7:187-95, 1993; U.S. Pat. No. 5,185,444, 1997. The oligomers werepurified by ion exchange chromatography and analyzed for purity byhigh-performance liquid chromatography (HPLC) and mass spectrometry. Theamount of full-length product was generally >90%. Before use, they wereprepared as concentrated stock solutions with distilled water and storedat 4° C.

Cell Culture

Cells were obtained from the American Type Culture Collection (ATCC,Rockville, Md.) or were derived in this laboratory. They were culturedin a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham'snutrient mixture F-12 supplemented with glutamine (2 mM), streptomycin(100:g/ml), and penicillin (100 U/ml). Dialyzed fetal bovine serum (FBS)was purchased from either Sigma (St. Louis, Mo.) or Hyclone (Ogden,Utah). WI-38 and HeLa cells were cultured in 10% serum and NRK cellswere cultured in 4% serum.

Recombinant Plasmids and Cell Lines

The human myc genomic clone pHSR-1 was a deposit from M. Bishop to theATCC. The 2.2-kilobase (kb) 5′-end was adapted for cloning into aluciferase vector by PCR using appropriate restriction sitesincorporated into the primers (Scharf, 1990). The luciferase vector wasadapted from one supplied by Clontech Inc. (Palo Alto, Calif.) to allowN-terminal fusion proteins with insect luciferase. Plasmids wereintroduced into HeLa cells using the Lipofectin protocol and reagentfrom Life Science Technologies (Gaithersburg, Md.) using the neomycingene/Geneticin selection procedure (F Colbere-Garapin et al., J. Mol.Biol. 150:1 -14, 1981). The rabbit α-globin-luciferaseconstruct-containing cell line has been described (M Partridge et al.,Antisense & Nucleic Acid Drug Dev. 6:169-75, 1996).

[³H] Thymidine Incorporation Assay

Cells were trypsinized, counted, and plated at 400,000 cells/2 ml/wellinto 6-well dishes. On day 2, PMO antisense agents were added to thedesired concentration, and the cells were scrape loaded (P L McNeil etal., J. Cell Biol. 98:1556-64, 1984, Partridge et al., cited above) witha rubber policeman using a gentle sweeping motion. The cell suspensionwas pipetted once to partly disaggregate any clumps formed, and 1 ml wastransferred to a 24-well dish containing an additional 1 ml/well offresh medium. For NRK cells, on day 4, 1 μCi of [³H]thymidine (DuPont,NEN, Wilmington, Del.) (NET-027) was added per well, and 6 hours later,the cells were washed twice with phosphate-buffered saline (PBS),precipitated with 5% trichloroacetic acid, washed a further two timeswith PBS, solubilized with 0.2 N NaOH/0.1% SDS, and the amount ofradioactivity was incorporated into DNA quantitated in a scintillationcounter. The quantitation procedure was identical for WI-38 cells exceptthat the incubation period for incorporation was 15 hours. In general,each agent or concentration was assayed in duplicate, and the valueswere averaged. The duplicates were usually within 10% of each other.

Luciferase Assay

The myc-luciferase HeLa cells were scrape loaded in a manner similar tothe normal fibroblasts with the following changes. One million cellswere plated into 6-well dishes, and on day 2 the entire 2 ml volume wastransferred to another 6-well dish. Thirty hours later, the cells werecollected and assayed for luciferase light production as described(Partridge et al.; 1996, Summerton et al., 1997; cited above).

Cell Cycle Analysis

Cells were analyzed for cell cycle stage by flow cytometry. The cellswere scrape loaded as described, and two wells were combined andreplated in 10-cm dishes to obtain enough cells for analysis byfluorescence-activated cell sorting (FACS). Two days later, the cellswere trypsinized, washed with PBS and resuspended in cold 80% ethanolfor at least 2 hours. Following the fixation step, the cells werecollected by centrifugation and stained with the DNA fluorochromepropidium iodide (Telford et al., cited above). The ethanol-treatedpellet was resuspended in 1 ml of 1 mM EDTA, 50:g/ml propidium iodide,1:l/ml Triton X-100, and 10:g/ml RNase A. After at least 1 hour atambient temperature, the cell suspension was analyzed using a CoulterEpic XL-MCL flow cytometer (Coulter Electronics, Hialeah, FL) with anexciting wavelength of 488 nm. Data were analyzed using Phoenix Systems(San Jose, Calif.) multicycle program software.

mRNA Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis

To examine the effects of Morpholino oligomers on myc RNA splicing, HeLacells were plated in 6-well dishes at 1 million cells/well. The nextday, oligomers at 20:M final concentration were added to the medium andscrape loaded into the cells as detailed previously. Twenty-four hourslater, the loaded cells were harvested by trypsinization and RNAprepared. Two wells were combined for each sample. Cytoplasmic (mature)RNA was extracted from the cell pellet by a Triton X-100 lysis procedureusing a Qiagen Rneasy Mini Kit (Chartsworth, Calif.) following thedirections for “isolation of RNA from the cytoplasm of animal cells.”The RNA was eluted in 30 :1 of water with a yield of about 10-20:g.

Six microliters of RNA (2-3:g) was reverse transcribed in a final 20:1reaction mixture with 1×PCR buffer (10 mM Tris, pH 8.3, 50 mM KCl, 1.5mM MgCl₂) (Perkin-Elmer, Norwalk, Conn.) 1 mM of each deoxynucleotidetriphosphate, 0.75:g 9-mer random primers, and 250 U Moloney murineleukemia virus (MmuLV) RT (New England BioLabs, Beverly, Mass.). Afteraddition of enzyme, the reactions were incubated for 10 minutes at 25°C., following by 30 minutes at 42° C., and 4 minutes to denature thepolymerase at 94° C.

Exon 1-exon 2 PCR was performed using a two-step nested PCR procedure.Step 1 primers had the sequences 5′-CGG GCA CTT TGC ACT GAA ACT TAC AACACC (SEQ ID NO: 51) and 5′-GGT CGC AGA TGA AAC TCT GGT T (SEQ ID NO:52). One microgram of each of the primers was added to the 20:1 RTreaction, and the volume was adjusted to 100:1 with 1×PCR buffer. Fourunits of Amplitaq (Perkin-Elmer) Taq polymerase were added, and 30cycles were performed with steps of 94° C. for 30 seconds, 62° C. for 30seconds, and 72° C. for 40 seconds. Step 2 used primer sequences of5′-CTC CTT GCA GCT GCT TAG ACG CTG G (SEQ ID NO: 53) and 5′-GAA GGG TGTGAC CGC AAC GTA GGA G (SEQ ID NO: 54). The step 1 reaction mixture (4:1)was added to 96 :1 of 1×PCR buffer with 200 nM of each triphosphate and1 μg of each primer. Step 2 PCR conditions were 30 cycles of 94° C. for30 seconds, 68° C. for 40 seconds, and 74° C. for 30 seconds. Aliquotswere then analyzed by agarose gel electrophoresis.

Although the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention. Sequence Table SEQ ID Genbank NO Sequence (5′→3′) Target^(‡)Acc. No. Location  1 CTGTGCTTAC/CGGGTTTTCCACCTCCC Rat c-myc (SD) Y00396 2553-79  2 ATCGTCGTGACTGT/CTGTTGGAGGG Rat c-myc (SA)  4140-64  3GCTCACGTTGAGGGGCATCG Rat c-myc  4161-80 (ds of SA)  4GGTCACTCAC/CGGTAGAGAA Rat CYP3A2 (SD) X62087  1155-74  5GGGTTCCAAGT/CTATAAAGG Rat CYP3A2 (SA)  1526-45  6* TGTGTCTTTTCCAG Humanandrogen M35845    31-44 receptor exon 2  7* TTTGGAGACTGCCAGGGACCATGHuman androgen    45-67 receptor exon 2  8 CATGGTCCCTGGCAGTCTCC Humanandrogen    48-67 receptor exon 2  9 TCAATGGGCAAAACATGGTCCCTGGCAGT Humanandrogen    45-80 CTCCAAA receptor exon 2 10* TTTGTGTTCTCCCAG Humanandrogen M35846    28-43 receptor exon 3 11* GGAAACAGAAGTACCTGTGCGCCHuman androgen    44-66 receptor exon 3 12 GGCGCACAGGTACTTCTG Humanandrogen    49-66 receptor exon 3 13 AATCATTTCTGCTGGCGCACAGGTACTTC Humanandrogen    44-79 TGTTTCC receptor exon 3 14 CCCCTGCAGCACGCGGGT HumanHCG-B X00266  1321-38 subunit 15 GAGGCAGGGCCGGCAGGACCCCCTGCAGC HumanHCG-B  1321-57 ACGCGCGT subunit 16 GGCATCGTCGCGGGAGGCTG Human c-mycJ00120  4506-25 17 GGGCATCGTCGCGGGAGGCT ″  4507-26 18GGGGCATCGTCGCGGGAGGC ″  4508-27 19 AGGGGCATCGTCGCGGGAGG ″  4509-28 20GAGGGGCATCGTCGCGGGAG ″  4510-29 21 TGAGGGGCATCGTCGCGGGA ″  4511-30 22TTGAGGGGCATCGTCGCGGG ″  4512-31 23 GTTGAGGGGCATCGTCGCGG ″  4513-32 24CGTTGAGGGGCATCGTCGCG ″  4514-33 25 ACGTTGAGGGGCATCGTCGC ″  4515-34 26AACGTTGAGGGGCATCGTCG ″  4516-35 27 TAACGTTGAGGGGCATCGTC ″  4517-36 28CTAACGTTGAGGGGCATCGT ″  4518-37 29 GCTAACGTTGAGGGGCATCG ″  4519-38 30AGCTAACGTTGAGGGGCATC ″  4520-39 31 AAGCTAACGTTGAGGGGCAT ″  4521-40 32GAAGCTAACGTTGAGGGGCA ″  4522-41 33 TCCTCATCTTCTTGTTCCTC ″  6656-75 34AACAACATCGATTTCTTCCTCATCTTCTT ″  6656-91 GTTCCTC 35 CCCGGAAGGCAGTCTGGCHuman p53 X54156 11691-708 36 TCCTCCATGGCAGTGACCCGGAAGGCAGT ″ 11689-724CTGGCTG 37 CTACTGGCCGCTGAAGGGC Human ab1 (ds AJ131466   376-94 ofbcr-ab1 fusion point) 38 GCTCAAAGTCAGATGCTACTGGCCGCTGA Human ab1 (ds  374-409 AGGGCTT of bcr-ab1 fusion point) 39TCGTCGGTCTCTCCGCTTCTTCTTGCC HIV-1 rev U69590  5517-43 (prior art) 40CTCTGGTGGTGGGTAAGGGT HIV-1 rev L39106  7885-7904 41CGGGTCTGTCGGGTTCCCTCTGGTGGTGG ″  7885-7921 GTAAGGGT 42GGGGCAUCGUCGUGACUGU/CUGUUGGAGGG Rat c-myc (SA) Y00396  4140-69 43CGUCGUGACUGU/CUGUUGGAGG ″ Y00396  4141-62 44 CGTCGTGACTGT/CTGTTGGAGG ″Y00396  4141-62 45 GGCAUCGUCGCGGGAGGCUG/CUGGAGCG Human c-myc (SA) J00120 4498-4505 46 CCGCGACAUAGGACGGAGAGCAGAGCCC Rat c-myc Y00396  4364-91 47ACTGTGAGGGCGATCGCTGC derived from SEQ (scrambled) ID NO: 25 48ACGATGAGTGGCATAGTCGC derived from SEQ (3 mismatches) ID NO: 25 49CTCCGCAATGCTGAAAGGTG Rat BCL-2 (cntrl) 50 GGCGUGCCUCAAACAUGGUGGCGG RatPCNA-1 (cntrl) 51 CGGGCACTTTGCACTGAAACTTACAACACC primer sequence 52GGTCGCAGATGAAACTCTGGTT ″ 53 CTCCTTGCAGCTGCTTAGACGCTGG ″ 54GAAGGGTGTGACCGCAACGTAGGAG ″*native sequence, not antisense^(‡)unless otherwise indicated, antisense target is downstream (ds) ofsplice acceptor (SA) junction; SD = splice donor junction

1. An antisense compound characterized by: (a1) a morpholino backbonecomposed of morpholino subunits that are linked by unchargedphosphorodiamidate linkages interspersed with at least two and up tohalf positively charged phosphorodiamidate linkages, (a2): abase-sequence length of between 12 and 25 nucleotide bases; and (a3): abase sequence that is complementary to a target region of a selectedpreprocessed mRNA coding for a selected protein, where the 5′ end of thetarget region is 1-25 bases downstream of a normal splice acceptor sitein said preprocessed mRNA, and having the properties that: (b1): thecompound is taken up by eukaryotic cells; (b2): the compound hybridizesto the target region of preprocessed mRNA in such cells, and (b3): thecompound so hybridized to the target pre-mRNA prevents splicing at saidacceptor splice site, such that the splice mechanism proceeds to adownstream splice acceptor site in the pre-mRNA, producing a splicevariant processed mRNA with a truncated coding sequence.
 2. Thecomposition of claim 1, wherein said morpholino subunits are linked byphosphorodiamidate linkages, in accordance with the structure:

where Y₁═O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X for the uncharged linkages is alkyl, alkoxy,thioalkoxy, or an alkyl amino of the form wherein NR₂, where each R isindependently hydrogen or methyl, and for the positively chargedlinkages, X is 1-piperazine.
 3. The compound of claim 1, wherein the 5′end of the target region is 2-20 bases downstream of said normal spliceacceptor site.
 4. The compound of claim 3, wherein the 5′ end of thetarget region is 2-15 bases downstream of said normal splice acceptorsite.
 5. The compound of claim 1, wherein said downstream spliceacceptor site is a whole multiple of three bases downstream of thenormal splice acceptor site, such that said splice variant mRNA has acoding sequence in frame with that of the processed mRNA when it isnormally spliced.
 6. The compound of claim 1, wherein said selectedprotein has multiple distinct binding regions, and said truncated codingsequence codes for a variant protein in which one such binding region isdisabled.
 7. The compound of claim 1, wherein said variant protein is adominant negative protein.
 8. The compound of claim 7, wherein saidselected protein is human c-myc, and said variant protein is anN-terminal truncated c-myc.
 9. The compound of claim 8, having a basesequence selected from the group consisting of SEQ ID NO: 3 and SEQ IDNOs: 13 through
 28. 10. The compound of claim 1, wherein the selectedprotein and corresponding antisense base sequence is selected from thegroup consisting of (a) human chorionic gonadotropin, β subunit: SEQ IDNO: 6; (b) human androgen receptor: SEQ ID NO: 9 or 12; (c) human c-myc:SEQ ID NO: 3, 13, or 29; (d) human p53: SEQ ID NO: 30; (e) human abl:SEQ ID NO: 31; and (f) HIV-1 rev: SEQ ID NO:
 33. 11. A method ofinhibiting normal splicing of mRNA in a eukaryotic cell, comprisingcontacting the cell with the an antisense compound characterized by:(a1) a morpholino backbone composed of morpholino subunits that arelinked by uncharged phosphorodiamidate linkages interspersed with atleast two and up to half positively charged phosphorodiamidate linkages(a2): a base-sequence length of between 12 and 25 nucleotide bases; and(a3): a base sequence that is complementary to a target region of aselected preprocessed mRNA coding for a selected protein, where the 5′end of the target region is 1-25 bases downstream of the a normal spliceacceptor site in said preprocessed mRNA, wherein the compound: (b1): istaken up by the cell; (b2): hybridizes to the target region ofpreprocessed mRNA in the cell, and (b3): being so hybridized, (i)prevents splicing at said normal acceptor splice site, such that (ii)the splice mechanism proceeds to a downstream splice acceptor sequencein the mRNA, producing a splice variant processed mRNA with a truncatedcoding sequence.
 12. The method of claim 11, wherein said morpholinosubunits are joined by phosphorodiamidate linkages, in accordance withthe structure:

where Y₁═O, Z=O, Pj is a purine or pyrimidine base-pairing moietyeffective to bind, by base-specific hydrogen bonding, to a base in apolynucleotide, and X for the uncharged linkages is alkyl, alkoxy,thioalkoxy, or an alkyl amino of the form wherein NR₂, where each R isindependently hydrogen or methyl, and for the positively chargedlinkages, X is 1-piperazine.
 13. The method of claim 11, wherein the 5′end of the target region is 2-20 bases downstream of said normal spliceacceptor site.
 14. The compound of claim 13, wherein the 5′ end of thetarget region is 2-15 bases downstream of said normal splice acceptorsite.
 15. The method of claim 11, wherein said downstream spliceacceptor site is a whole multiple of three bases downstream of thenormal splice acceptor site, such that said splice variant mRNA has acoding sequence in frame with that of the processed mRNA when it isnormally spliced.
 16. The method of claim 11, wherein the selectedprotein has multiple distinct binding regions, and said truncated codingsequence codes for a variant protein in which a binding region isdisabled.
 17. The method of claim 16, wherein said variant protein is adominant negative protein.
 18. The method of claim 17, wherein saidselected protein is human c-myc, and said variant protein is anN-terminal truncated c-myc.
 19. The method of claim 18, wherein theantisense compound has a base sequence selected from the groupconsisting of SEQ ID NO: 3 and SEQ ID NOs: 13 through
 28. 20. The methodof claim 11, wherein the selected protein and corresponding antisensebase sequence is selected from the group consisting of (a) humanchorionic gonadotropin, β subunit: SEQ ID NO: 6; (b) human androgenreceptor: SEQ ID NO: 9 or 12; (c) human c-myc: SEQ ID NO: 3, 13, or 29;(d) human p53: SEQ ID NO: 30; (e) human abl: SEQ ID NO: 31; and (f)HIV-1 rev: SEQ ID NO: 33.